Twenty-one of the results were validated using traditional individual luciferase transfection experiments. The variant and reference allele binding sites of the selected mirSNPs were individually cloned into the 3′ UTR of the luciferase gene within the pIS-0 plasmid and transfected into HEK293, HepG2, and HeLa cells. This included seven of the miRNA target sequences, each with reference and variant sequences, tested in three cell lines for a total of 21 validations. Within each cell line, the luciferase activity in the cells transfected with the reference plasmid was compared to the activity in the cells transfected with the variant plasmids. The effect of the variants in these individual luciferase assays were compared with the results from the PASSPORT-seq assay (Figure 4). In 17 of the 21 comparisons, the statistical significance of the results and the direction of the effect of the variant matched the PASSPORT-seq results (Supplementary Figure 8 and Supplementary Table 3). In an additional two comparisons (rs3134615 in HeLa and HEK293 cells), the results were statistically significant in one assay, but not the other, but the direction and magnitude
